Transgenic plant of the species solanum tuberosum with resistance to phytophthora

ABSTRACT

The present invention concerns a transgenic plant of the species  Solanum tuberosum  with a resistance to an oomycete of the genus  Phytophthora , transgenic parts of such a plant, a method for its manufacture and to a composition for external application to plants. On the one hand, nucleotide sequences in accordance with SEQ ID NOS:1-43 are provided from  Phytophthora  in a host plant-induced gene silencing strategy in potato plants; on the other hand, a fungicide for plant treatment is provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application under 35 U.S.C.§371 of International Patent Application PCT/DE2013/000446 filed Aug. 6,2013 which claims priority to German Patent Application 10 2012 016009.7 filed Aug. 8, 2012. The International Application was published onFeb. 13, 2014, as International Publication No. WO 2014/023285 under PCTArticle 21(2). The entire contents of these applications are herebyincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 15, 2015, isnamed 245761.000009_SL.txt and is 100,249 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates to a transgenic plant of the speciesSolanum tuberosum with a resistance to an oomycete of the genusPhytophthora, to transgenic parts a plant of this type, to a method forits manufacture and to a means for external application to plants.

Even now, potato late blight caused by Phytophthora infestans is stillthe most prevalent and most economically important potato disease.

Throughout the globe, the pathogen results in loss of earnings, withharvest losses of more than 20 percent. This means that expensivechemical plant protection means have to be used, because the naturaldefence mechanisms of the potato with the help of which P. infestans iscombatted or with which propagation can be slowed down and restricted isnot sufficient or not permanent.

Natural plant defence mechanisms, such as the hypersensitive reaction atthe infection site, lignification of the cell wall, the production of PR(pathogenesis-related) proteins and the synthesis of phytoalexins areindeed known to contribute to augmenting resistance, but they are alwaysaccompanied by an energy loss and thus a loss of earnings for affectedplants.

Natural defence mechanisms in plants also include the expression ofso-called resistance genes (R genes), the gene products of whichinteract with microbial avirulence genes (Avr genes) (gene for genehypothesis) and thus induce a specific defence reaction. This resistancecan, however, be interrupted if a pathogen such as P. infestans candispense with the synthesis of the Avr gene and recognition of thepathogen and thus the subsequent specific defence reaction in the planthost does not occur.

Fire et al. (1998) have already demonstrated that double stranded RNA(dsRNA) can result in the sequence-specific degradation of homologousRNA. Starting from these results, transgenic plants have been developedin the meantime which, with the aid of RNA interference (RNAi) by meansof host plant-induced silencing of conserved and essential genes, forexample from nematodes or Lepidoptera- and Coleroptera species, canexhibit resistance to these pests in vitro as well as in vivo.

In addition, the host plant-phytopathogenic fungus interaction canconstitute an application of the concept of host-induced gene silencing(HIGS) to induce resistance (EP 1 716 238).

Van West et al. (1999) initially used the gene silencing method inPhytophthora, in order to carry out functional analyses of theseoomycete-specific genes.

In WO 2006/070227, the use of RNA interference to control fungalpathogens based on contact of dsRNA with fungal cells outside the fungalcell was described for the first time. It proposes a method for themanufacture of a pathogen-resistant plant. In this manner, the RNAinterference can be directed against one or more genes of a pathogen aswell as several pathogens. Phytophthora infestans is mentioned as apossible fungal pathogen and potato as a possible host plant.

Previous studies have given rise to the hypothesis that hostplant-induced gene silencing does not work for every gene and choice ofthe target gene is essential for functional silencing. Thus, forexample, the plasma membrane H+-ATPase PnMA1 in Phytophthora parasiticacould not be reduced sufficiently by host plant-induced gene silencingto deliver efficient protection against a pathogen (Zhang et al. 2011).According to this, selection of the target genes is also decisive foreffective pathogen defence (Yin et al. 2011).

Recently, a screening system was proposed which was supposed tofacilitate the selection of suitable parasitic genes for silencingconstructs for the production of pathogen-resistant plants (US2010/0257634). The identification of appropriate test constructs toinduce phytoresistance in potato was also proposed by the authors. Inthis regard, target genes were defined based on bioinformatic analysesof genome sequences or based on sequence homologies to essential genesor virulence factors from known model organisms. That document does notcontain any indications of the genes disclosed in the present inventionfor the generation of a resistance against an oomycete of the genusPhytophthora.

A method for producing a broad spectrum resistance in transgenic plantsagainst multiple fungi is described in WO 2009/112270. In oneimplementation of the method of that invention, the broad spectrumresistance is directed against Uncinula necator, Plasmopora viticola,Uromyces spec., Phakopsora pachyrhizi, Erysiphe sp. and also P.infestans.

Furthermore, the development of Phytophthora infestans-resistant potatoplants through RNAi-induced silencing is disclosed in WO 2006/047495. Onthe one hand, plants were generated which carry gene sequences of therRNA gene from Phytophthora infestans for RNA interference. Thesilencing construct described in WO 2006/047495 directed against therRNA gene of Phytophthora infestans comprises base pairs 1-600 ofAccession number AJ854293 and with it 32 bp of the coding region of the18S rRNA as well as the complete coding region of the 5.8 S rRNA gene ofthe blight pathogen. When selecting the target genes for HIGSstrategies, with a view to applicability, it is vital that it has asshort as possible or preferably no homologies extending over more than17 sequential base pairs to the gene sequences of non-target organisms,as if there were, gene expression of the non-target organisms in thecase of consumption of the transgenic plant or its harvest product couldbe destroyed (“off-target” effect). However, the sequence described inin WO 2006/047495 comprises 32 bp of the P. infestans 18S rRNA, whichhas 100% identity with the homologous sequence of the 18S rRNA gene fromman (Homo sapiens), pigs (Sus scrofa) and cattle (Bos taurus). Humanpotato consumption in Asia in 2005 was 26 kg, in North America it was 58kg and in Europe it was 96 kg per person (FAOSTAT). In the light of thehigh human and animal consumption of potatoes, the rRNA sequences fromPhytophthora infestans described in WO 2006/047495 as HIGS target genesare unsuitable for consumers on safety grounds.

On the other hand, in WO 2006/047495, plants were produced that carrygene sequences for the cathepsin B gene from Myzus persicae and theelicitin gene INF1 from P. infestans for RNA interference and thusexhibit resistance to two plant pathogens. The target gene INF1 usedtherein codes for an elicitor. A resistance based on an elicitor as apathogenicity factor is a disadvantage because the elicitin gene INF1 isnot always necessary for an infection of potatoes by Phytophthorainfestans (Kamoun et al. 1998).

BRIEF SUMMARY OF THE INVENTION

The aim of the present invention is thus to provide a transgenic plantof the species Solanum tuberosum which is pathogen-resistant to anoomycete of the genus Phytophthora and in particular is suitable forconsumption.

In accordance with the invention, this aim is accomplished by the factthat a double-stranded first and second DNA are stably integrated intothe transgenic plant, wherein the first DNA comprises (a) a nucleotidesequence in accordance with SEQ ID NOS: 1-43, or (b) a fragment of atleast 15 successive nucleotides of a nucleotide sequence in accordancewith SEQ ID NOS: 1-43, or (c) a nucleotide sequence which iscomplementary to one of the nucleotide sequences of (a) or (b), or (d) anucleotide sequence which hybridizes with one of the nucleotidesequences of (a), (b) or (c) under stringent conditions.

Surprisingly, it has been found that a first DNA of this type isparticularly suitable for conferring a pathogen resistance in potatoplants via a host-induced gene silencing strategy.

The first and the second DNA are integrated in a stable manner into thegenome of a transgenic plant of the species Solanum tuberosum.Preferably, the DNAs are integrated in a stable manner into a chromosomeof the plant. However, they can also be integrated into anextra-chromosomal element. The advantage of stable integration is thatthe DNA can be passed on to subsequent generations of the transgenicplant.

The double-stranded DNA is composed of a coding and a non-coding strand.

Furthermore, the nucleotide sequence of the coding strand of the secondDNA is the reverse complement of the nucleotide sequence of the codingstrand of the first DNA. The term “reverse complement” with respect to anucleotide sequence in the 5′-3′ direction should be understood to meana nucleotide sequence in the 3′-5′ direction wherein, in accordance withthe base pairing rules, the bases correspond to the bases of the firstDNA and are in a reverse/mirrored sequence. If the nucleotide sequenceof the coding strand of the first DNA is atggttc, for example, then thereverse complementary nucleotide sequence of the coding strand of thesecond DNA is gaaccat. This is also known as sense and correspondingantisense (reverse complementary) orientation of the nucleotidesequences.

In particular, the nucleotide sequence of the coding strand of thesecond DNA can be the reverse complement of the nucleotide sequence ofthe coding strand of the first DNA over the whole length of thesequence. However, it can also be only partially reverse complementary,i.e. reverse complementary over a limited length. The nucleotidesequence of the coding strand of the second DNA can also be reversecomplementary in more than one region, for example in two or threeregions of its nucleotide sequence to the nucleotide sequence of thecoding strand of the first DNA.

Starting from the completely or partially reverse complementarynucleotide sequences for the coding strand of the first and second DNA,a double-stranded RNA is produced. The double-stranded structure of theRNA arises by the formation of bridging hydrogen bonds betweencomplementary nucleotides. Double-stranded RNA regions may be formedover a single nucleic acid strand which is partially complementary toitself, or over two different, discontinuous complementary nucleic acidstrands. The bridging hydrogen bond formation may thus be intramolecularas well as intermolecular.

In accordance with the invention, the first DNA comprises a nucleotidesequence in accordance with SEQ ID NOS: 1-43, wherein these sequencesare nucleotide sequences from selected target genes from Phytophthorainfestans. The group formed by these target genes comprises essentialgenes for primary metabolism as well as for amino acid synthesis, inparticular the biosynthesis of aliphatic amino acids (valine, leucine,isoleucine) as well as for glutamate biosynthesis, genes for cellregulation and signal transduction as well as redox regulation, calciumsignalling, G-protein signalling, MAP-kinase signalling andtranscription factors, as well as genes for translation components, genewith RNA processing functions, genes which code for developmental anddifferentiation proteins such as, for example, with cell wall formationfunctions, as well as genes which code for transporters, channelling andmembrane proteins. A summary of these target genes from Phytophthorawhich are used for designing the host-induced gene silencing is set outin Table 1.

TABLE 1 Target identifi- ID gene_ID Function Category cation 1PITG_03410 Acetolactate synthase Amino acid biosynthesis A 2 PITG_00375Haustorium-specific mem- Development/ D brane protein (Pihmp1)differentiation 3 PITG_13490 Urokanase Glutamate biosynthesis C 4PITG_00146 Glucose-6-P-dehydrogenase Primary metabolism C 5 PITG_00561Ubiquinone- biosynthesis Primary metabolism B protein COQ9 6 PITG_06732Acyl-CoA-dehydrogenase Primary metabolism B 7 PITG_07405 Pyruvate kinasePrimary metabolism B 8 PITG_12228 NADH-cytochrome B5 Primary metabolismB reductase 9 PITG_15476 Malate dehydrogenase Primary metabolism B 10PITG_18076 Phosphoglycerate mutase Primary metabolism B 11 PITG_19736Alcohol dehydrogenase Primary metabolism B 12 PITG_20129/Acyl-CoA-dehydrogenase Primary metabolism B 13 PITG_00221 Tryptophansynthase Amino acid biosynthesis A 14 PITG_05318 N-(5′-phosphoribosyl)Amino acid biosynthesis C anthranilate-isomerase 15 PITG_13139 Threoninesynthase Amino acid biosynthesis C 16 PITG_00578 Imidazolone propionaseGlutamate biosynthesis C 17 PITG_15100 Histidine ammonium lyaseGlutamate biosynthesis A 18 PITG_11044 Protein phosphatase Signaltransduction B 19 PITG_21987 Protein phosphatase 2C Signal transductionB 20 PITG_01957 Calcineurin-like catalytic Calcium signalling C subunitA 21 PITG_02011 Calcineurin-subunit B Calcium signalling C 22 PITG_16326Calcineurin-like catalytic Calcium signalling C subunit A 23 PITG_00708Thioredoxin Redox regulation C 24 PITG_00715 Thioredoxin Redoxregulation C 25 PITG_00716 Thioredoxin Redox regulation C 26 PITG_09348Glutaredoxin Redox regulation C 27 PITG_08393 PsGPR11 G-protein coupledG-Protein signalling D receptor 28 PITG_10447 SAPK homologue MAP Kinasesignalling D 29 PITG_06748 Myb-like DNA-binding protein Transcriptionfactor A 30 PITG_19177 C2H2-transcription factor Transcription factor D(PsCZF1-homologue) 31 PITG_06873 Aspartyl-tRNA-synthetase Translation B32 PITG_09442 40S Ribosomal protein S21 Translation B 33 PITG_16015Ribonuclease RNA-processing B 34 PITG_09306 PnMas2- homologueDevelopment/ D differentiation 35 PITG_03335 Callose synthase (Fks1/2-Cell wall formation D homologue) 36 PITG_05079 Glycosyl transferase(Fks1/2- Cell wall formation D Homologue) 37 PITG_18356 Beta-glucanesynthesis-associated Cell wall formation D protein (KRE6-homologue) 38PITG_09193 Aquaporin Channel B 39 PITG_00562 Mitochondrialtricarboxylate Transporter B carrier 40 PITG_08314 ABC superfamilyprotein Transporter B 41 PITG_12289 ATPase H- or Na-translocatingTransporter B F-type 42 PITG_12999 MFS superfamily transporterTransporter B 43 PITG_16478 Acyl-CoA-dehydrogenase Primary metabolism B

DETAILED DESCRIPTION OF THE INVENTION

The term “gene silencing” or silencing describes processes for switchinggenes off. Silencing can, for example, be transcriptional orpost-transcriptional. Gene silencing also includes antisense technology,RNAi, or dsRNA.

The expression of a nucleotide sequence of a target gene in Phytophthorainfestans is selectively inhibited by gene silencing. A targetnucleotide sequence can in this case also be a non-processed RNAmolecule, an mRNA or a ribosomal RNA sequence.

The target genes were identified by (i) publically available expressionstudies such as microarray data regarding oomycete differentiation orinfection processes, for example, and publically available data on theinvestigation of metabolic processes during oomycete differentiation orinfection (Grenville-Briggs et al. 2005, Judelson et al. 2009a, Judelsonet al. 2009b) (A), (ii) comparative bioinformatic studies coupled withpedantic analysis (BioMax Bioinformatic Framework) (B), (iii) analysesof metabolic pathways coupled with pedantic analysis (C) as well as (iv)evaluations of publically available data regarding the characterizationof homologous genes in eukaryotic organisms (Roemer et al. 1994, Inoueet al. 1995, Mazur et al. 1995, Lesage et al. 2004, Avrova et al. 2008,Wang et al. 2009, Li et al. 2010, Wang et al. 2010) (D).

When selecting the target genes, care was taken that the nucleotidesequence of these genes was specific for P. infestans in order toexclude unwanted silencing of plant and human genes. To this end, theselected target genes were compared as regards their proteins (BlastX)with the proteome of Solanum tuberosum and Solanum lycopersicum. At thesame time, the target gene sequences were compared as regards theirnucleotides (BlastN) with the genome of Solanum tuberosum, Solanumlycopersicum and a general BlastN (criteria: BlastN; database: humangenomic+transcript; optimize for: somewhat similar sequences (blastn)).Target genes were considered to be highly suitable when they exhibitedno nucleotide homologies with Solanum tuberosum and Solanum lycopersicumand no or only partial homologies in general BlastN in only shortsequence regions (<17 nts), so that an interaction with endogenous plantnucleotide sequences was inhibited or did not occur.

In accordance with the invention, the nucleotide sequences used may havedifferent lengths. Thus, the nucleotide sequences of one of SEQ ID NOS:1-43 may, for example, have a length of between 501 and 735 nucleotides.

The nucleotide sequences used may also be one or more fragments of oneor more nucleotide sequences of SEQ ID NOS: 1-43. In this regard, thefragments comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200 or 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400 or 2500successive nucleotides of one or more of the nucleotide sequences of SEQID NOS: 1-43. A particularly suitable fragment is a fragment of thenucleotide sequence of SEQ ID NO: 1 with 290 nucleotides.

In a preferred embodiment of the invention, combinations of two, three,four, five, six, seven, eight, nine, ten or more fragments of the samenucleotide sequence as that of SEQ ID NO: 1 or different nucleotidesequences such as those of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43 are used. Apreferred combination comprises fragments of the nucleotide sequences ofSEQ ID NOS: 4, 23, 27 and 28, the genes of which are involved withsignal transduction. A further preferred combination comprises fragmentsof nucleotide sequences of SEQ ID NOS: 3, 16 and 17; these are genes forglutamate biosynthesis from P. infestans. Further advantageouscombinations comprise nucleotide sequences or fragments of nucleotidesequences from genes for cell wall formation (SEQ ID NOS: 25, 36, 37),calcium signalling (SEQ ID NOS: 20, 21, 22), primary metabolism genes(SEQ ID NOS: 5, 6, 7), redox regulation genes (SEQ ID NOS: 24, 25, 26),or comprise several nucleotide sequences or fragments of nucleotidesequences from transporter genes in accordance with SEQ ID NOS: 39, 40,41, 42. Further preferred combinations comprise nucleotide sequences orfragments of nucleotide sequences of different target gene groups suchas, for example, genes for G-protein signalling, MAP kinase signalling,primary metabolism and redox regulation (SEQ ID NOS: 27, 28, 4, 23).Combining several target genes means that the possibility that theresistance of the transgenic plant could be disrupted by a naturalmutation in the oomycete is avoided.

The double-stranded first DNA introduced into the potato plant of theinvention may comprise a nucleotide sequence which hybridizes understringent conditions with one of the following nucleotide sequences: (a)a nucleotide sequence in accordance with SEQ ID NOS: 1-43, or (b) afragment of at least 15 successive nucleotides of a nucleotide sequencein accordance with SEQ ID NOS: 1-43, or (c) a nucleotide sequence whichis complementary to one of the nucleotide sequences of (a) or (b), or(c). Examples of stringent conditions are: hybridizing in 4×SSC at 65°C. and then washing several times in 0.1×SSC at 65° C. for approximately1 hour in total. The term “stringent hybridization conditions” as usedhere can also mean: hybridization at 68° C. in 0.25 M sodium phosphate,pH 7.2 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequentlywashing twice with 2×SSC and 0.1% SDS at 68° C.

The present invention also in particular encompasses such fragments ofnucleotide sequences which have a few, for example 1 or 2 nucleotides,which are not complementary to the target gene sequence fromPhytophthora infestans. Sequence variations which, for example, occur inoomycetes, which are based on a genetic mutation, for example byaddition, deletion or substitution or a polymorphism in a Phytophthorainfestans strain and which result in wrong pairing over a region of 1, 2or more nucleotides, can be tolerated as long as the RNA formed by thetransgenic potato plant can still interfere with the target gene RNAformed by the oomycete.

In accordance with the invention, the transgenic plant of the speciesSolanum tuberosum confers a pathogen resistance to an oomycete of thegenus Phytophthora. To determine the resistance, the transgenic potatoplant is compared with a control plant which ideally has the identicalgenotype to the transgenic plant and has been grown under identicalconditions, but which does not contain the DNA which has been introducedinto the transgenic plant. The resistance can be determined using anoptical score, wherein scores of 0 (not susceptible) to 100 (verysusceptible) are awarded. Preferably, the transgenic plants of theinvention confer a resistance which, compared with a control plant,results in a reduced propagation of infection over the plant surface ofat least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100 percent (see the conditions under “measuring the resistancein transgenic potato plants under outdoor conditions).

The genus Phytophthora comprises various species, for example thespecies alni, cactorum, capsici, cinnamomi, citrophthora, clandestina,fragariae, hedraiandra, idaei, infestans, ipomoeae, iranica, kernoviae,mirabilis, megakarya, nicotianae, palmivora, parasitica, phaseoli,ramorum, pseuodotsugae, quercina, sojae or tentaculata.

In a preferred embodiment of the invention, the transgenic potato plantexhibits a resistance to Phytophthora infestans.

Already the inhibition of the biosynthesis of the aliphatic amino acidsvaline, leucine and isoleucine by a construct directed againstacetolactate synthase from P. infestans results in a substantialreduction in blight infestation and a drastic increase in leafresistance under laboratory and field conditions. By combining severaltarget genes such as, for example, genes from G protein signalling, MAPkinase signalling, primary metabolism, amino acid biosynthesis and redoxregulation in one construct, the resistance effect can be increasedstill further, since the pathogen is inhibited by the multiple action ofthe combination construct on the use of alternative signalling andmetabolic pathways.

Surprisingly, the defensive power of the potato plants of the inventionagainst isolates of P. infestans of varying aggressivity is changed in amanner such that a resistance is obtained which protects the plantsefficiently and permanently against this most important of pathogens.

It was also surprisingly discovered that the resistance has no greatenergetic disadvantages or negative changes in the agronomic propertiesof the potato plant. The cultivation trials under near-field conditionsdid not have any deleterious effects on the quality of the plants.Careful selection of the target genes of P. infestans which excludes anyhomology extending over 17 successive base pairs with genes fromnon-target organisms (potato, human, pig, cattle) means that there areno restrictions on using the plants as a feed or foodstuff and norestrictions as regards sowing, cultivation, harvesting or processingthe crop. The plants can freely be used as agricultural, food or feedplants.

In accordance with a preferred embodiment, the double-stranded RNA ismiRNA or siRNA. MiRNA describes small interfering RNA and includesnaturally produced miRNAs and synthetic miRNAs which, for example, canbe produced by recombinant or chemical synthesis or by processingprimary miRNA.

SiRNA describes small interfering RNA and includes naturally producedsiRNAs and synthetic siRNAs which, for example, can be produced byrecombinant or chemical synthesis or by processing dsRNAs.

The transgenic plant produces dsRNA from the introduced double-strandedDNA which is processed by endogenous RNAi or silencing mechanisms toform siRNAs and miRNAs.

In order to obtain dsRNA, a double-stranded first DNA with a nucleotidesequence in accordance with one of SEQ ID NOS: 1-43 or a fragmentthereof in the sense orientation and a double-stranded second DNA in theantisense orientation can be used which are separated by an intron whichhas no similarity with the target genes in question. As an example, theDNA may be orientated with a nucleotide sequence of SEQ ID NO: 1 againstthe acetolactate synthase gene from Phytophthora infestans. Uponexpression in a plant cell, an RNA transcript is formed which, becauseof the homology between the sense and antisense sequence regions, cancoalesce to form a dsRNA. Because the missing base pairs in the regionof the intron, the dsRNA forms a hairpin structure. A dsRNA with ahairpin structure can also be prepared by means of one double-strandedDNA with a nucleotide sequence in accordance with one of SEQ ID NOS:1-43 in the sense orientation and a second in the antisense orientationwith a different length. In this respect, the nucleotide sequence in thesense orientation may be about 190 nucleotides longer than thenucleotide sequence in the antisense orientation, or vice versa.

Defined sequence regions for the selected nucleotide sequences of thetarget genes are amplified by PCR and cloned both in the sense and inthe antisense direction into a vector which is suitable for thesynthesis of hairpin structures. In this regard, several fragments withsequence regions of different target genes can be cloned into a vectorin order to construct a combination hairpin construct. The vectors canbe introduced into a plant cell using transformation methods which areknown in plant biotechnology. The skilled person will be aware that, forexample, a selected nucleotide sequence of a target gene can also becloned into one vector in the sense orientation and the nucleotidesequence of the target gene can be cloned into a second vector in theantisense orientation and then introduced into a plant cell byco-transformation, for example.

The silencing mechanism arises from dsRNA such as, for example, hairpinRNA structures or gene duplexes. The dsRNA will produce small dsRNAs bymeans of a dsRNA-specific endonuclease (dicer), which are processed bymeans of longer nucleotide sequences into small dsRNAs preferably of21-25 base pairs, a process which is similar for both “stem-loop”(primary miRNA) and also for long complementary dsRNA precursors.Argonaut proteins, as central components of the RNA-induced silencingcomplexes (RISC), bind and unwind siRNA and miRNA so that the leadstrand of the duplex binds specifically by base pairing to the mRNA andleads to its degradation. By means of miRNA, RNAi behaves in acomparatively similar process, with the difference that the miRNAproduced also comprises partial regions which are not identical to thetarget genes.

After infestation of a host plant with Phytophthora infestans, anexchange of RNA formed in the plant which is directed against one ormore Phytophthora-specific target sequences can occur between the hostplant and the oomycetes. In the oomycetes, these RNAs can lead tosequence-specific gene silencing of one or more target genes. Proteinsand protein complexes such as dicers, RISC (RNA-induced silencingcomplex) as well as RNA-dependent RNA polymerase (RdRP), can participatein this process.

The siRNA effect is known to be continued in plants when the RdRPsynthesises new siRNAs from the degraded mRNA fragments. This secondaryor transitive RNAi can reinforce silencing and also result in silencingof different transcripts when they share these highly conservedsequences.

In a preferred embodiment, the first DNA and the second DNA areoperatively linked with at least one promoter.

A “promoter” is a non-translated DNA sequence, typically upstream of acoding region which contains the binding site for the RNA polymerase andinitiates transcription of the DNA. A promoter contains special elementswhich function as regulators for gene expression (for examplecis-regulatory elements). The term “operatively linked” means that theDNA which comprises the integrated nucleotide sequence is linked to apromoter in a manner such that it allows expression of this nucleotidesequence. The integrated nucleotide sequence may be linked with aterminator signal downstream as a further component.

The promoter can be of plant, animal or microbial origin, or it may beof synthetic origin and can, for example, be selected from one of thefollowing groups of promoters: constitutive, inducible,development-specific, cell type-specific, tissue-specific ororganospecific. While constitutive promoters are active under mostconditions, inducible promoters exhibit expression as a result of aninducing signal which, for example, may be issued by biotic stressorssuch as pathogens or abiotic stressors such as cold or dryness orchemicals.

Examples of promoters are the constitutive CaMV 35S promoter (Benfey etal., 1990) as well as the C1 promoter which is active in green tissue(Stahl et al., 2004).

The first and second DNA may also, however, be operatively linked to adouble promoter such as, for example, the bidirectionally active TR1′and TR2′ promoter (Saito et al., 1991).

Furthermore, the first and the second DNA may each be operatively linkedto a promoter.

The use of two promoters, which each flank the 3′ end and the 5′ end ofthe nucleic acid molecule, enables expression of the respectiveindividual DNA strand, wherein two complementary RNAs are formed whichhybridize and form a dsRNA. In addition, the two promoters can bedeployed such that one promoter is directed towards the transcription ofa selected nucleotide sequence and the second promoter is directedtowards the transcription of a nucleotide sequence which iscomplementary to the first nucleotide sequence. As long as bothnucleotide sequences are transcribed, a dsRNA is formed.

Further, a bidirectional promoter can be deployed which allows theexpression of two nucleotide sequences in two directions, wherein onenucleotide sequence is read off in the 3′ direction and a secondnucleotide sequence is read off in the 5′ direction. As long as bothnucleotide sequences are complementary to each other, a dsRNA can beformed.

The present invention also concerns parts of a transgenic plant of thespecies Solanum tuberosum.

In the context of this application, the term “parts” of the transgenicplant in particular means seeds, roots, leaves, flowers as well as cellsof the plant of the invention. In this regard, the term “cells” shouldbe understood to mean, for example, isolated cells with a cell wall oraggregates thereof, or protoplasts. “Transgenic parts” of the transgenicplant also means those which can be harvested, such as potato tubers,for example.

Furthermore, the present invention concerns a method for the manufactureof a transgenic plant of the species Solanum tuberosum which exhibits aresistance against an oomycete of the genus Phytophthora.

Suitable methods for the transformation of plant cells are known inplant biotechnology. Each of these methods can be used to insert aselected nucleic acid, preferably in a vector, into a plant cell inorder to obtain a transgenic plant in accordance with the presentinvention. Transformation methods can include direct or indirect methodsfor transformation and can be used for dicotyledenous plants andprimarily also for monocotyledenous plants. Suitable directtransformation methods include PEG-induced DNA uptake, liposome-inducedtransformation, biolistic methods by means of particle bombardment,electroporation or microinjection. Examples of indirect methods areagrobacterium-induced transformation techniques or viral infection bymeans of viral vectors.

A preferred method which is employed is agrobacterium-induced DNAtransfer using binary vectors. After transformation of the plant cells,the cells are selected on one or more markers which were transformed inthe plant with the DNA of the invention and comprise genes whichpreferably induce antibiotic resistance such as, for example, theneomycin phosphotransferase II gene NPTII, which induces kanamycinresistance, or the hygromycin phosphotransferase II gene HPTII, whichinduces hygromycin resistance.

Next, the transformed cells are regenerated into complete plants. AfterDNA transfer and regeneration, the plants obtained may, for example, beexamined by quantitative PCR for the presence of the DNA of theinvention. Resistance tests on these plants against Phytophthorainfestans in vitro and in the greenhouse are next. Routine furtherphenotypic investigations can be carried out by appropriately trainedpersonnel in the greenhouse or outdoors. These transformed plants underinvestigation can be cultivated directly.

The method of the invention for the manufacture of a transgenic plant ofthe species Solanum tuberosum which exhibit a resistance against anoomycete of the genus Phytophthora comprises the following steps:

(i) producing a transformed first parent plant containing adouble-stranded first DNA which is stably integrated into the genome ofthe parent plant and which comprises (a) a nucleotide sequence inaccordance with SEQ ID NOS: 1-43, or (b) a fragment of at least 15successive nucleotides of a nucleotide sequence in accordance with SEQID NOS: 1-43, or (c) a nucleotide sequence which is complementary to oneof the nucleotide sequences of (a) or (b), or (d) a nucleotide sequencewhich hybridizes with one of the nucleotide sequences of (a), (b) or (c)under stringent conditions;(ii) producing a transformed second parent plant containing adouble-stranded second DNA which is stably integrated into the genome ofthe parent plant, wherein the nucleotide sequences for the coding strandof the first and second DNA are partially or completely reversecomplementary with respect to each other;(iii) crossing the first parent plant with the second parent plant;(iv) selecting a plant in the genome of which a double-stranded firstDNA and a double-stranded second DNA has been stably integrated in orderto confer a pathogen resistance against an oomycete of the genusPhytophthora so that a double-stranded RNA can be produced therefrom.

In accordance with the invention, it is a nucleotide sequence or afragment of a nucleotide sequence in accordance with SEQ ID NOS: 1-43from Phytophthora infestans.

In a preferred embodiment of the invention, the double-stranded RNA canbe miRNA or siRNA.

The invention also concerns a composition for external application toplants.

This composition is prepared for external application to plants. Itcontains double-stranded RNA, wherein one strand of this RNA correspondsto the transcript of a double-stranded DNA comprising (a) a nucleotidesequence in accordance with SEQ ID NOS: 1-43, or (b) a fragment of atleast 15 successive nucleotides of a nucleotide sequence in accordancewith SEQ ID NOS: 1-43, or (c) a nucleotide sequence which iscomplementary to one of the nucleotide sequences of (a) or (b), or (d) anucleotide sequence which hybridizes with one of the nucleotidesequences of (a), (b) or (c) under stringent conditions.

Double-stranded RNA for the manufacture of the composition in accordancewith the invention can be produced in vitro using methods known to theskilled person. As an example, the double-stranded RNA can besynthesized by forming the RNA directly in vitro. The double-strandedRNA can also be synthesized from a double-stranded DNA by formation ofan mRNA transcript which then forms a hairpin structure, for example.

The composition in accordance with the invention can be used as afungicide for a plant or its seed. In this regard, the composition isused to control the growth of the pathogen, for containing thepropagation of the pathogen or for the treatment of infected plants. Asan example, the composition can be used as a fungicide for spraying inthe form of a spray, or other routine ways which are familiar to theskilled person for external application to the plant tissue or byspraying or mixing with the cultivation substrate before or after theplants have sprouted.

In a further application, the composition in accordance with theinvention is used as a pre-treatment for seed. In this regard, thecomposition is initially mixed with a carrier substrate and applied tothe seeds in a combination which comprises the double-stranded RNA andthe carrier substrate, whereby the carrier substrate has anRNA-stabilizing effect, for example. Thus, the RNA stability and thusits action on the selected target genes of Phytophthora infestans can beincreased, for example by chemical modifications such as the exchange ofribose for a hexose. Liposomes which encapsulate the RNA molecules canalso be used as RNA stabilizers.

Ideally, the plants treated with the composisiton are those of thespecies Solanum tuberosum.

The discussion above regarding the plant of the invention and the methodof the invention also apply to this composition.

The present invention will now be described with reference to thefigures and sequences:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Plasmid pRNAi as an exemplary representation of a vector whichcan be used for the formation of hairpin constructs against a targetgene. This vector contains a CaMV 35S promoter, a multiple cloning site,an intron from the gene AtAAP6 which codes for an amino acid permease inArabidopsis thaliana, a further multiple cloning site as well as a CaMV35S terminator.

FIG. 2: Plasmid pRNAi_PITG_(—)03410 as an exemplary representation of avector which contains a sense-intron-antisense fragment for theformation of dsRNA against a target gene (here PITG_(—)03410). Thisvector additionally contains a CaMV 35S promoter, a multiple cloningsite, an intron from the gene AtAAP6 which codes for an amino acidpermease in Arabidopsis thaliana, a further multiple cloning site aswell as a CaMV 35S terminator.

FIG. 3: Plasmid pRNAi_HIGS_CoA as an exemplary representation of avector which contains various defined sequences in thesense-intron-antisense fragment, which should lead to the formation ofdsRNA against various target genes. This vector additionally contains aCaMV 35S promoter, a multiple cloning site, an intron from the geneAtAAP6 which codes for an amino acid permease in Arabidopsis thaliana, afurther multiple cloning site as well as a CaMV 35S terminator.

FIG. 4: Plasmid pGBTV/EcoRI_kan. Binary Ti plasmid which was used as acloning vector.

FIG. 5: Plasmid pGBTV/EcoRI_kan_PITG_(—)03410. Binary Ti plasmid whichwas used for agrobacterium-induced transformation.

FIG. 6: Plasmid pAM, which was used as a cloning vector.

FIG. 7: Plasmid pAM_HIGS_CoA, as an example of a plasmid which was usedas a cloning vector.

FIG. 8: Plasmid p95P-Nos. Binary Ti plasmid which was used as a cloningvector.

FIG. 9: Plasmid p95N_HIGS_CoA. Binary Ti plasmid which was used foragrobacterium-induced transformation.

FIG. 10: Plasmid p95N_HIGS_dPRNAi_PITG_(—)03410 as an exemplaryrepresentation of a binary vector for the formation of dsRNA against atarget gene (here PITG_(—)03410) using two CaMV 35S promoters which eachflank the 3′- and the 5′ end of the nucleic acid molecule.

FIG. 11: Transgenic potato shoot on selection medium aftertransformation in the regeneration stage.

FIG. 12: Diagnostic PCR for testing the transgenicity of potatoes(PR-H4) after transformation with the binary vectorpGBTV/EcoRI_kan_PITG_(—)03410.

Detection of sense fragment (370 bp) (primer S3345′-ATCCCACTATCCTTCGCAAG-3′ (SEQ ID NO: 44)×S12595′-TTGATATCGCGGAAGGCGAGAGACATCG-3′ (SEQ ID NO: 45)) and antisensefragment (450 bp) (S 329 5′-CTAAGGGTTTCTTATATGCTCAAC-3′ (SEQ ID NO:46)×S1259 5′-TTGATATCGCGGAAGGCGAGAGACATCG-3′ (SEQ ID NO: 45)). Mix:PCR-MasterMix, PCR monitoring. Marker: Tracklt™ 1 Kb DNA Ladder.

FIG. 13 A: Detection of siRNAs in transgenic potato plants aftertransformation with the binary vector pGBTV/EcoRI_kan_PITG_(—)03410.Detection was carried out by hybridization of the Northern Blot with theradioactively labelled probe dsRNA_. Multiple applications of varioussamples from the lines PR-H4_T007 and TO11. FIG. 13 B: Detection ofsiRNAs in transgenic potato plants after transformation with the binaryvector p95N HIGS_PITG_(—)00375. Detection was carried out byhybridization of the Northern Blot with the radioactively labelled probedsRNA_PITG00375. Single application of the samples from the linesPR-H2_T040, T045, T047 and T049.

FIG. 14 A: Plasmid pABM-70Sluci_dsRNA.PITG_(—)00375 as an exemplaryrepresentation of a vector which contains a fusion construct consistingof the luciferase reporter gene and the test HIGS target fragmentPITG_(—)00375. The vector additionally contains a double CaMV 35Spromoter, a multiple cloning site, the coding sequence for the luc genefrom Photinus pyralis, which codes for a luciferase, separated from amodified intron PIV2 from the potato gene St-LS1 (Eckes et al. 1986,Vancanneyt et al. 1990), a further multiple cloning site as well as aNos terminator from the nopalin synthase gene from Agrobacteriumtumefaciens.

FIG. 14 B: Plasmid pABM-70Sluci_dsRNA.PITG_(—)03410 as an exemplaryrepresentation of a vector which contains a fusion construct consistingof a luciferase reporter gene and the test HIGS target gene fragmentPITG_(—)03410.

FIG. 15 A: Relative luciferase activity in transgenic potato lines ofthe genotype Baltica with stable integration of the HIGS_RNAi constructagainst the PITG_(—)03410 gene from P. infestans after bombardment withthe vector pABM-70Sluci_dsRNA.PITG_(—)03410. B: Baltica (non-transgeniccontrol), T003, T005 transgenic HIGS potato lines.

FIG. 15 B: Relative sporangia production from P. infestans on transgenicHIGS lines. The potato lines of the variety Baltica were transformedwith an RNAi construct in order to form dsRNA against the P. infestansgene PITG_(—)03410. After infection with P. infestans in the detachedleaf assay, these lines exhibited a reduced sporangia productioncompared with the non-transgenic variety Baltica (mean of 4 biologicalrepetitions). B: Baltica (non-transgenic control), T003, T005:transgenic HIGS potato lines.

FIG. 16 A: Relative Luciferase activity in transgenic potato lines ofthe genotype Hermes with stable integration of the HIGS_RNAi constructagainst the PITG_(—)03410 gene from P. infestans after bombardment withthe vector pABM-70Sluci_dsRNA.PITG_(—)03410. H: Hermes (non-transgeniccontrol), T004, TO11: transgenic HIGS potato lines.

FIG. 16 B: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Hermes were transformed withan RNAi construct in order to form dsRNA against the P. infestans genePITG_(—)03410. After infection with P. infestans in the detached leafassay, these lines exhibited a reduced sporangia production comparedwith the non-transgenic variety Baltica (mean of 4 biologicalrepetitions). H: Hermes (non-transgenic control), T004, TO11: transgenicHIGS potato lines.

FIG. 17 A: Relative luciferase activity in transgenic potato lines ofthe genotype Desiree with stable integration of the HIGS_RNAi constructagainst the PITG_(—)03410 gene from P. infestans after bombardment withthe vector pABM-70Sluci_dsRNA.PITG_(—)03410. D: Desiree (non-transgeniccontrol), T098: transgenic HIGS potato line.

FIG. 17 B: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Desiree were transformedwith an RNAi construct in order to form dsRNA against the P. infestansgene PITG_(—)03410. After infection with P. infestans in the detachedleaf assay, these lines exhibited a reduced sporangia productioncompared with the non-transgenic variety Baltica (mean of 4 biologicalrepetitions). D: Desiree, (non-transgenic control), T098: transgenicHIGS potato line.

FIG. 18 A: Relative Luciferase activity in transgenic potato lines ofthe genotype Desiree with stable integration of the HIGS_RNAi constructagainst the PITG_(—)00375 gene from P. infestans after bombardment withthe vector pABM-70Sluci_dsRNA.PITG_(—)00375. D: Desiree, (non-transgeniccontrol), T042, T044 T047, T049: transgenic HIGS potato lines.

FIG. 18 B: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Desiree were transformedwith a RNAi construct in order to form dsRNA against the P. infestansgene PITG_(—)00375. After infection with P. infestans in the detachedleaf assay, these lines exhibited a reduced sporangia productioncompared with the non-transgenic variety Baltica (mean of 4 biologicalrepetitions). D: Desirée, (non-transgenic control), T042, T044 T047,T049: transgenic HIGS potato lines.

FIG. 19 A: Level of infection in transgenic potato lines of the genotypeHermes with stable integration of the HIGS_RNAi construct against thePITG_(—)03410 gene from P. infestans after infection of the plants underoutdoor-like conditions with P. infestans. Grey lines with triangle:Baltica, Desirée, and Russet Burbank (non-transgenic controls), Blacklines with square: plants of the genotype Hermes: solid line: Hermes(non-transgenic control), dashed line: PR-H-4-7 & dotted line:PR-H-4-11: transgenic HIGS potato lines.

FIG. 19 B: Photographic documentation of the degree of infection of thetransgenic potato lines PR-H-4-7 and PR-H-4-11 of the genotype Hermeswith stable integration of the HIGS_RNAi construct against thePITG_(—)03410 gene from P. infestans after infection of the plants underoutdoor-like conditions with P. infestans compared with thenon-transgenic control Hermes. Photographs taken 32 days post-infection.

FIG. 20: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Russet Burbank weretransformed with a RNAi construct in order to form dsRNA against the P.infestans gene PITG_(—)03410. After infection with P. infestans in thedetached leaf assay, these lines exhibited a reduced sporangiaproduction compared with the non-transgenic variety Russet Burbank (meanof 3 biological repetitions). Russet Burbank (non-transgenic control);H-4-T084, H-4-T096: transgenic HIGS potato lines.

FIG. 21: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Hermes were transformed witha RNAi construct in order to form dsRNA by means of a double promoterconstruct HIGS_dPRNAi_PITG_(—)03410 against the P. infestans genePITG_(—)03410. After infection with P. infestans in the detached leafassay, these lines exhibited a reduced sporangia production comparedwith the non-transgenic variety Hermes (mean of 3 biologicalrepetitions). Hermes (non-transgenic control); H-23-T0003, H-23-T026,H-23-T038, H-23-T062, H-23-T063, H-23-T066: transgenic HIGS potatolines.

FIG. 22: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Russet Burbank weretransformed with an RNAi construct HIGS_CoA in order to form dsRNAagainst the genes PITG_(—)00146, PITG_(—)08393, PITG_(—)10447 andPITG_(—)00708 from P. infestans. After infection with P. infestans inthe detached leaf assay, these lines exhibited a reduced sporangiaproduction compared with the non-transgenic variety Russet Burbank (meanof 3 biological repetitions). Russet Burbank (non-transgenic control);H-13-T050, H-13-T053, H-13-T036, H-13-T032: transgenic HIGS potatolines.

FIG. 23: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Russet Burbank weretransformed with a RNAi construct in order to form dsRNA against the P.infestans gene PITG_(—)06748. After infection with P. infestans in thedetached leaf assay, these lines exhibited a reduced sporangiaproduction compared with the non-transgenic variety Russet Burbank (meanof 3 biological repetitions). Russet Burbank (non-transgenic control);H-15-T008, H-15-T010: transgenic HIGS potato lines.

FIG. 24: Relative sporangia production of P. infestans on transgenicHIGS line. The potato line of the variety Russet Burbank weretransformed with an RNAi construct in order to form dsRNA against the P.infestans gene PITG_(—)09306. After infection with P. infestans in thedetached leaf assay, these lines exhibited a reduced sporangiaproduction compared with the non-transgenic variety Russet Burbank (meanof 3 biological repetitions). Russet Burbank (non-transgenic control);H-10-T111: transgenic HIGS potato line.

FIG. 25: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Russet Burbank weretransformed with an RNAi construct in order to form dsRNA against the P.infestans gene PITG_(—)09193. After infection with P. infestans, in thedetached leaf assay, these lines exhibited a reduced sporangiaproduction compared with the non-transgenic variety Russet Burbank (meanof 3 biological repetitions). Russet Burbank (non-transgenic control);H-12-T194, H-12-T195, H-12-T222, H-12-T239: transgenic HIGS potatolines.

FIG. 26: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Desirée were transformedwith an RNAi construct in order to form dsRNA against the P. infestansgene PITG_(—)09193. After infection with P. infestans, in the detachedleaf assay, these lines exhibited a reduced sporangia productioncompared with the non-transgenic variety Desirée (mean of 3 biologicalrepetitions). Desirée (non-transgenic control); H-12-T187, H-12-T216,H-12-T237, H-12-T245: transgenic HIGS potato lines.

FIG. 27: Relative sporangia production of P. infestans on transgenicHIGS lines. The potato lines of the variety Russet Burbank weretransformed with an RNAi construct in order to form dsRNA against the P.infestans gene PITG_(—)19177. After infection with P. infestans, in thedetached leaf assay, these lines exhibited a reduced sporangiaproduction compared with the non-transgenic variety Russet Burbank (meanof 3 biological repetitions). Russet Burbank (non-transgenic control);H-9-T271, H-9-T305, H-9-T308: transgenic HIGS potato lines.

FIG. 28: Relative sporangia production of P. infestans on transgenicHIGS line. The potato line of the variety Desiree were transformed withan RNAi construct in order to form dsRNA against the P. infestans genePITG_(—)19177. After infection with P. infestans in the detached leafassay, these lines exhibited a reduced sporangia production comparedwith the non-transgenic variety Desiree (mean of 3 biologicalrepetitions). Desiree (non-transgenic control); H-9-T280: transgenicHIGS potato line.

FIG. 29: Plasmid p95N_RNAi PITG_(—)06748 as an exemplary representationof a vector which contains a sense-intron-antisense fragment in order toform dsRNA against a target gene (here PITG_(—)06748). This vectoradditionally contains a CaMV 35S promoter, a multiple cloning site, anintron from the gene AtAAP6 which codes for an amino acid permease inArabidopsis thaliana, a further multiple cloning site as well as a CaMV35S terminator.

FIG. 30: Plasmid p95N_RNALPITG_(—)09306 as an exemplary representationof a vector which contains a sense-intron-antisense fragment in order toform dsRNA against a target gene (here PITG_(—)09306). This vectoradditionally contains a CaMV 35S promoter, a multiple cloning site, anintron from the gene AtAAP6 which codes for an amino acid permease inArabidopsis thaliana, a further multiple cloning site as well as a CaMV35S terminator.

FIG. 31: Plasmid p95N_RNALPITG_(—)09193 as an exemplary representationof a vector which contains a sense-intron-antisense fragment in order toform dsRNA against a target gene (here PITG_(—)09193). This vectoradditionally contains a CaMV 35S promoter, a multiple cloning site, anintron from the gene AtAAP6 which codes for an amino acid permease inArabidopsis thaliana, a further multiple cloning site as well as a CaMV35S terminator.

FIG. 32: Plasmid p95N_RNALPITG_(—)19177 as an exemplary representationof a vector which contains a sense-intron-antisense fragment in order toform dsRNA against a target gene (here PITG_(—)19177). This vectoradditionally contains a CaMV 35S promoter, a multiple cloning site, anintron from the gene AtAAP6 which codes for an amino acid permease inArabidopsis thaliana, a further multiple cloning site as well as a CaMV35S terminator.

EXEMPLARY EMBODIMENTS Preparation of Constructs

Defined sequence regions of the selected target genes were amplifiedusing PCR and cloned in both the sense and the antisense direction intoa pRNAi vector which is suitable for the system of hairpin structures(FIG. 2). In this manner, several fragments with sequence regions fromvarious target genes can be cloned into a vector in order to generate acombination hairpin construct (FIG. 3).

Starting from genomic DNA from Phytophthora infestans, a sequence regionof 290 bp from the coding region of the gene PITG_(—)03410 was amplifiedusing PCR, cleaved via the restriction enzyme cleaving sites XhoI andSmaI inserted via the primer sequences and cloned into the pRNAi vector(primer 1: cgctcgaggctggatctcgcgctgaggt (SEQ ID NO: 47), primer 2:ttgatatcgcggaaggcgagagacatcg (SEQ ID NO: 45)). This vector contains aCaMV 35S promoter, a multiple cloning site, an intron from the geneAtAAP6 which codes for an amino acid permease in Arabidopsis thaliana, afurther multiple cloning site as well as a CaMV 35S terminator. This wascleaved with XhoI and Ec1136II and the 4.098 kb vector fraction wasseparated using agarose gel electrophoresis and then isolated. Theligation solution was transformed in E. coli strain XL1-blue(Stratagene, LaJolla, Calif.). The same PITG_(—)03410 fragment was thencloned into the plasmid pRNAi_PITG_(—)03410_sense in the antisensedirection. To this end, the fragment was again amplified from genomicDNA from Phytophthora infestans using PCR, cleaved via the restrictionenzyme cleaving sites XhoI and SmaI inserted via the primer sequencesand then ligated into the vector pRNAi_PITG_(—)03410_sense which hadbeen cleaved with SmaI-SaII and then linearized (FIG. 1). Thesense-intron-antisense (RNAi-PITG_(—)03410) gene fragment was cleavedout of the pRNAi vector and cloned into the vector pGBTV/EcoRI_kan (FIG.4). To this end, both pGBTV/EcoRI_kan and pRNAi_PITG_(—)03410 werecleaved with HindIII and ligated so that the plasmidpGBTV/EcoRI_kan_PITG_(—)03410 was generated (FIG. 5). Alternatively,HIGS-RNAi constructs such as HIGS-CoA were initially cloned into thevector pAM (DNA Cloning Service e.K., Hamburg) (FIG. 6). To this end,both pAM and pRNAi_PITG_(—)03410 were cleaved with HindIII and ligated,so that the plasmid pAM_HIGS_CoA was generated (FIG. 7). From the vectorpAM, the HIGS_CoA fragment was integrated into the vector p95P-Nos (DNACloning Service e.K., Hamburg) by Sfil digestion and ligation (FIG. 8),so that the plasmid p95N_HIGS_CoA was generated (FIG. 9), which was usedfor potato transformation.

Alternatively to a vector as described above which is suitable for thesynthesis of hairpin structures, a section of the coding target generegion in the potato plant can be caused to carry out expression withthe aid of two oppositely (reverse) orientated promoters (FIG. 10). Inaddition, gene silencing can also be envisaged by means of artificialmicroRNA constructs (amiRNA) using the Web microRNA Designers (WMD3)protocol. Artificial miRNAs are 21-mer single stranded RNAs which can besynthesised in order to specifically negatively regulate desired genesin plants. Regulation happens—like with siRNAs—via mRNA cleavage. TheseRNAi constructs are then cloned into a binary vector and transformed byAgrobacterium tumefaciens-induced transformation in potatoes.

Transformation and Regeneration

Transformation of the potatoes was carried out in accordance with themodified protocol by Pel et al (2009) using the antibiotic kanamycin.The donor material was cultivated in 80 mL MS(D) (25° C.; 16 h day/8 hnight; 2000 lux) for 3-4 weeks. For transformation (C1), the internodeswere cut out of the donor material in approximately 0.5 cm explants.These were cultivated in petri dishes with 10 mL MS(D) (15-20Explants/dish) with 70 μl of an Agrobacterium tumefaciens culture whichhad been cultivated overnight at 28° C., which had earlier beentransformed with the HIGS-RNAi construct as part of a binary vector suchas, for example, p95N, incubated at 25° C. for 2 days in the dark. Next,the explants were dried on filter paper and placed in petri dishes onMSW-Medium with selection antibiotic (400 mg/L timentin+75 kanamycinmg/L) which were hermetically sealed and cultivated for 2 weeks (25° C.;16 h day/8 h night; 2000 lux) (C2). This selection step was repeatedevery 2 weeks until the shoots had regenerated (from C3). Regeneratedshoots (FIG. 11) were incubated on MS (30 g/L saccharose) with selectionantibiotic (250 timentin mg/L+100 kanamycin mg/L) to cause rooting andtested by PCR for integration of the construct to be transformed andthus for the presence of the nucleic acids of the invention. The use ofthe primers Bo2299 (5′-GTGGAGAGGCTATTCGGTA-3′ (SEQ ID NO: 48)) andBo2300 (5′-CCACCATGATATTCGGCAAG-3′ (SEQ ID NO: 49)) led to theamplification of a 553 bp DNA fragment from the bacterial NPTII gene,which codes for neomycin phosphotransferase. Furthermore, the sense andthe antisense fragment were detected using PCR, in order to ensure thatthe construct was complete (FIG. 12). The PCR was carried out using 10ng of genomic DNA, a primer concentration of 0.2 μM at an annealingtemperature of 55° C. in Multicycler PTC-200 (MJ Research, Watertown,USA). Propagation of the shoots which tested positive in the PCR wascarried out on MS+30 g/L saccharose+400 mg/L ampicillin.

Detection of Processed Double-Stranded RNA and siRNAs

In the transformed plants, the expressed hairpin or double-stranded RNAswere processed over the natural plant RNAi mechanisms in a manner suchthat these RNA molecules were degraded into small single stranded RNAs.These siRNAs are deposited on the mRNA of the corresponding target genein oomycetes and thus effect silencing of this gene. The plants are thusplaced in the position of protecting themselves against attackingpathogens. By means of this concept, transgenic potato plants can beproduced which have an increased resistance to P. infestans.

The transformation of potato plants with constructs for the expressionof hairpin or double-stranded RNAs should result in the fact that theresulting dsRNAs are processed to siRNAs in preference to the naturalplant RNAi mechanisms. In order to measure the fragmentation of thedsRNA, whole RNA was isolated from the transgenic plants using thetrizol method (Chomczynski and Sacchi, 1987). 15 μg of whole RNA/samplewas supplemented with formamide, denatured and separatedelectrophoretically in a 1% agarose gel with 10% formaldehyde in 1×MOPSbuffer (0.2 M MOPS (sodium salt), 0.05 M NaOAc, 0.01 M EDTA in DEPCdH₂O. pH 7.0 with NaOH). The separated RNA was transferred from the gelonto a nylon membrane (positively charged) using the Northern Blotmethod into 20×SSC buffer (saline-sodium citrate buffer). This washybridized with a radioactively labelled probe which was complementaryto the sequence of the target gene fragment which was present in thesense or in the antisense direction in the construct transformed intothe plants. In this manner, RNA fragments which are complementary to thesequence of the dsRNA fragment are labelled and detected by means of aphosphoimager.

The transformation of potato plants with constructs for the expressionof hairpin or double-stranded RNAs should result in the fact that theresulting dsRNA are processed to siRNAs in preference to the naturalplant RNAi mechanisms. In order to measure the fragmentation of thedsRNA into siRNAs, whole RNA was isolated from the transgenic plantsusing the trizol method (Chomczynski and Sacchi, 1987). 15 μg of wholeRNA/sample was supplemented with formamide, denatured and separatedelectrophoretically in a polyacrylamide gel with 15% Tris/boricacid/EDTA (TBE) and uric acid in 0.5×TBE. The separated RNA wastransferred onto a nylon membrane (neutral) from the gel using the TankBlot method in 0.5×TBE. This was hybridized with a radioactivelylabelled probe which was complementary to the sequence of the targetgene fragment which was present in the sense or in the antisensedirection in the construct transformed in the plants. In this manner,siRNAs which are complementary to the sections of sequence of the dsRNAfragment are labelled and detected by means of a phosphoimager.

In various transgenic potato lines such as, for example, PR-H4 lines orPR-H2 lines, such siRNAs could be detected (FIG. 13 A, B). This showsthat the constructs transformed in the plants are recognized andprocessed by plant RNAi mechanisms such that siRNAs against HIGS targetgenes from P. infestans can be formed which should carry out silencingof this gene in the pathogen.

Measurement of Resistance in Transgenic Potato Plants in the DetachedLeaf Assay

To test the resistance of the transgenic potato leaves, the transgenicplants were cultivated from in vitro plants in the greenhouse in 5 Lpots. After 6-8 weeks, 2 pinnae per plant were cut off and placed in asealed plastic box on a moist Grodan pad such that the leaf stem was inthe moist Grodan material. This ensured that the humidity was high. Theboxes were incubated at 18° C. using a day/night program (sunlight,February-September). The pinna leaflets were inoculated with drops of azoospore suspension (10 μL; 10⁴ zoospores/mL) of Phytophthora infestans.After 24 hours, the lid of the boxes was opened somewhat in order toallow a gentle circulation of air in the boxes. The optical scoring andquantification of the zoospores was carried out after 6 days. Theoptical scoring evaluated the degree of infection and the destruction ofthe pinna leaf by P. infestans. Counting the sporangia led toquantification of the reproductive ability of the pathogen in the plant.In this regard, the previously infected leaves of a pinna were incubatedin 5 mL of water in Falcon tubes on a shaker for 2 h, so that thesporangia were loosened from the leaf. The sporangia were then countedwith the aid of a Thoma counting chamber under a microscope.

In various HIGS potato lines which were generated by transformation ofvarious potato genotypes, a reduced sporangia count could be determinedafter infection with P. infestans (6 dpi) (FIG. 15B-18B). This indicatesthat the reproductive ability of the pathogen on the transgenic plantshas been restricted.

The gene PITG_(—)03410 was tested as a target gene for HIGS in thegenetic background of the potato varieties Baltica, Hermes and Desiréeas well as the variety Russet Burbank using a vector as shown in FIG. 5.The detached leaf assay applied to these transgenic plants of thevariety Russet Burbank showed that the reproductive ability of thepathogen on the transgenic plants was restricted compared withnon-transgenic control plants (FIG. 20).

As an alternative to vectors which are suitable for the synthesis ofhairpin structures, a vector as shown in FIG. 10 with the target genePITG_(—)03410 was introduced into potato plants of the variety Hermesand the transgenic lines were investigated in the detached leaf assay(FIG. 21). Here again, it was shown that the reproductive ability of P.infestans was limited on the transgenic plants compared withnon-transgenic control plants.

Potato plants of the variety Russet Burbank which had been transformedwith a combination vector against the genes PITG_(—)00146,PITG_(—)00708, PITG_(—)10447 and PITG_(—)08363 of FIG. 3 were alsotested in the detached leaf assay. It was observed that the reproductiveability of P. infestans on these transgenic plants was restrictedcompared with non-transgenic control plants (FIG. 22).

Similarly, the detached leaf assay showed that the reproductive abilityof P. infestans on transgenic plants of the variety Russet Burbank isrestricted when transformed with a vector in accordance with FIG. 29,FIG. 30, FIG. 31 or FIG. 32 which is directed against the genesPITG_(—)06748 (FIG. 23), PITG_(—)09306 (FIG. 24), PITG_(—)09193 (FIG.25) or PITG_(—)19177 (FIG. 27).

Even on transgenic plants of the variety Desirée which were transformedwith a vector as shown in FIG. 31 or FIG. 32 which is directed againstthe gene PITG_(—)09193 (FIG. 25) or PITG_(—)19177 (FIG. 27), thedetached leaf assay showed that the reproductive ability of P. infestanswas restricted compared to non-transgenic control plants.

Transient Test System for RNAi Vectors in Potato Leaves

A transient test system in accordance with Birch et al. (2010) wasdeveloped to investigate the functionality of the RNAi vectors againstselected target gene sequences of P. infestans. By means ofco-bombardment, a RNAi vector targeting a target gene was expressedtransiently in potato leaves together with a fusion construct consistingof a luciferase reporter gene and the test target fragment. If the dsRNAconstruct is processed in the RNAi vector, then the formation of dsRNAand the resulting formation of siRNAs should be ensured. These siRNAsshould not only carry out the degradation of the target gene fragmenttranscript, but also give rise to the fused reporter gene transcript, sothat with a functional RNAi construct, a reduction in the luciferaseactivity can be observed. The plasmid pABM-70Sluci comprises a doubleCaMV 35S promoter, a multiple cloning site, the coding sequence for theluc gene from Photinus pyralis, which codes for a luciferase, separatedfrom a modified intron PIV2 from the potato gene St-LS1 (Eckes et al.1986, Vancanneyt et al. 1990), a further multiple cloning site as wellas a Nos terminator from the nopalin synthase gene from Agrobacteriumtumefaciens. The PCR-amplified fragment of the coding sequence region,for example of the PITG_(—)03410 gene, was cloned into this plasmidpABM-70Sluci, which was also cloned into the pRNAi vector to produce thedsRNA construct (FIG. 14A).

This transient test system can not only be used for validation of thegeneral functionality of the RNAi construct, but also be used in orderto investigate different sequence regions of a gene as regards itssilencing effect and finally can select the best sequence regions of agene for optimal silencing. In addition, the bombardment of transgenicplants stably transformed with a RNAi construct can serve to determinethe silencing efficiency of the individual transgenic HIGS potato lineswhich, for example, can differ substantially depending on theintegration site for the construct. In various transgenic HIGS potatolines which are obtained by transformations in various potato genotypes,and which show a reduced sporangia count after infection with P.infestans, a reduction in the luciferase activity could also be measured(FIG. 15A-18A). This shows the functionality of the HIGS constructsprocessed to siRNAs in relation to a silencing of the target genesequence in the transgenic plants.

When the coding gene sequences which are to be silenced by a combinationconstruct such as pRNAi_HIGS-CoA, for example (target genes:PITG_(—)08393, PITG_(—)00146, PITG_(—)10447, PITG_(—)00708), each clonedinto the vector pABM_(—)70 Sluci behind the coding sequence for the lucgene from Photinus pyralis (pABM_(—)70 Sluci_PITG_(—)08393, pABM_(—)70Sluci_PITG_(—)00146, pABM_(—)70 Sluci_PITG_(—)10447, pABM_(—)70Sluci_PITG_(—)00708) and together with the vector pRNAi_HIGS_CoA, are tobe transiently expressed in potato leaves, the silencing efficiency ofthe combination construct can be analysed on the various target genes.This is also possible by the bombardment of transgenic plants stablytransformed with the RNAi combination construct with the individualfusion constructs consisting of the luciferase reporter gene and thetest coding sequences of the various target genes.

The luciferase activity determinations were carried out with the aid ofDual Luciferase® Reporter Assays (Promega, Mannheim) (Schmidt et al.2004).

Measurement of Resistance in Transgenic Potato Plants Under OutdoorConditions

For the resistance test for the transgenic potato plants under outdoorconditions, the transgenic plants were initially cultivated early in theyear (March) from in vitro plants in the greenhouse for 3 weeks inmultiport pads. Next, these plants were planted out into a greenhousewith a wire mesh roof in natural soil so that the plants were exposed toenvironmental conditions, for example temperature, sunlight,precipitation and humidity, which were comparable with field conditions.The plants were planted out in 3 plots each with 6 plants. After 8weeks, one pinna from each of 2 plants in a plot was inoculated with P.infestans by spray inoculation (750 μL; 10⁴ zoospores/mL). Plastic bagswere placed over these pinnae to ensure that the humidity would be highand to promote infection. After two days, these plastic bags wereremoved. Proliferation of the blight by Phytophthora infestans in thegreenhouse was scored optically and documented photographically everyweek. The criteria for scoring the infection was initially only on theinfected pinna leaf (0: no infection, 1: slight infection (½ number ofinfected pinna leaves infected), 2: infection on more than ½ leaves of apinna, 3: infection on all leaves of the pinna) and then the spread ofthe infection to the plant and the whole plot (4: infection also extendsto some other leaves of the plant, 6: infection also extends to otherplants, 8: infection also extends substantially to other plants, 10: 10%of the plants infected/destroyed, 20: 20% of plants infected/destroyed,100: 100% of the plants infected/destroyed).

In various transgenic HIGS potato lines (PR-H-4-7, PR-H-4-11) which wereobtained by transformations in the potato genotype Hermes, a greatlyreduced degree of infection of these plants during the course ofinfection with Phytophthora infestans was determined compared with thetransformation genotype Hermes which had been cultivated, planted outand infected as the control exactly as with the transgenic plants. Thereduced degree of infection was initially reflected by a greatly reducedinfection capability of the pathogen on the inoculated pinnae (scores 21days post-infection: PR-H-4-7: 3.3; PR-H-4-11: 3.2; Hermes 7.6) and atlater times by a substantially reduced propagation ability of thepathogen to these plants (scores 32 days post-infection: PR-H-4-7: 26;PR-H-4-11: 15; Hermes: 80) (FIG. 19 A, B).

By means of the tests described, not only could processing of the HIGSconstruct in transgenic potato plants to siRNAs be demonstrated, butalso the functionality of these constructs in respect of silencing ofthe target gene sequence in these transgenic plants, and an increasedresistance of these plants to P. infestans could be quantified by areduced sporangia production of the pathogen on these host plants. Sincethe identification of functional HIGS target genes is not possiblewithout careful testing of their functionality, these analyses asdescribed in detail here are particularly suitable for defining geneswhich are effective in the HIGS construct and for generating resistantHIGS plants.

REFERENCES

-   Avrova A O, Boevink P C, Young V, Grenville-Briggs L J, van West P,    Birch P R, Whisson S C (2008) A novel Phytophthora infestans    haustorium-specific membrane protein is required for infection of    potato. Cell Microbiol. 10(11):2271-84.-   Birch R G, Shen B, Sawyer B J, Huttner E, Tucker W O, Betzner A    S (2010) Evaluation and application of a luciferase fusion system    for rapid in vivo analysis of RNAi targets and constructs in plants.    Plant Biotechnol J. May 1; 8(4):465-75. Epub 2010 Jan. 19-   Blackman L M, Arikawa M, Yamada S, Suzaki T, Hardham A R (2011)    Identification of a mastigoneme protein from Phytophthora    nicotianae. Protist. 162(1):100-14.-   Benfey, P. N., Ren, L., and Chua, N.-H. (1990). Combinatorial and    synergistic properties of CaMV 35S enhancer subdomains. EMBO J. (9),    1685-1696.-   Chomczynski P, Sacchi N. (1987) Single-step method of RNA isolation    by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal    Biochem. 162(1):156-9-   Eckes P., Rosahl S., Schell J., Willmitzer L. (1986) Isolation and    characterization of a light-inducible, organ-specific gene from    potato and analysis of its expression after tagging and transfer    into tobacco and potato shoots. Molecular and General Genetics    205 (1) 14-22, DOI: 10.1007/B F02428027-   Fire A, Xu S, Montgomery M K, Kostas S A, Driver S E, Mello    C C. (1998) Potent and specific genetic interference by    double-stranded RNA in Caenorhabditis elegans. Nature February 19;    391(6669):806-11-   Grenville-Briggs L J, Avrova A O, Bruce C R, Williams A, Whisson S    C, Birch P R, van West P (2005) Elevated amino acid biosynthesis in    Phytophthora infestans during appressorium formation and potato    infection. Fungal Genet Biol. 42(3):244-56.-   Inoue S B, Takewaki N, Takasuka T, Mio T, Adachi M, Fujii Y,    Miyamoto C, Arisawa M, Furuichi Y, Watanabe T (1995)    Characterization and gene cloning of 1,3-beta-D-glucan synthase from    Saccharomyces cerevisiae. Eur J Biochem 231(3):845-54.-   Judelson H S, Narayan R D, Ah-Fong A M, Kim K S (2009a) Gene    expression changes during asexual sporulation by the late blight    agent Phytophthora infestans occur in discrete temporal stages. Mol    Genet Genomics. 281(2):193-206.-   Judelson H S, Tani S, Narayan R D (2009b) Metabolic adaptation of    Phytophthora infestans during growth on leaves, tubers and    artificial media. Mol Plant Pathol. 10(6):843-55.-   Kamoun S, van West P, Vleeshouwers V G, de Groot K E, Govers F.    (1998). Resistance of nicotiana benthamiana to phytophthora    infestans is mediated by the recognition of the elicitor protein    INF1. Plant Cell. September; 10(9):1413-26.-   Lesage G, Sdicu A M, Ménard P, Shapiro J, Hussein S, Bussey H (2004)    Analysis of beta-1,3-glucan assembly in Saccharomyces cerevisiae    using a synthetic interaction network and altered sensitivity to    caspofungin. Genetics. May; 167(1):35-49-   Li A, Wang Y, Tao K, Dong S, Huang 0, Dai T, Zheng X, Wang Y (2010)    PsSAK1, a Stress-Activated MAP Kinase of Phytophthora sojae, Is    Required for Zoospore Viability and Infection of Soybean. Mol Plant    Microbe Interact. 23(8):1022-31.-   Mazur P, Morin N, Baginsky W, el-Sherbeini M, Clemas J A, Nielsen J    B, Foor F (1995) Differential expression and function of two    homologous subunits of yeast 1,3-beta-D-glucan synthase. Mol Cell    Biol. 15(10):5671-81.-   Pel M A, Foster S J, Park T H, Rietman H, van Arkel G, Jones J D G,    Van Eck H J, Jacobsen E, Visser R G F, Van der Vossen E A G (2009)    Mapping and cloning of late blight resistance genes from Solanum    venturii using an interspecific candidate gene approach. MPMI    22:601-615-   Roemer T, Paravicini G, Payton M A, Bussey H (1994) Characterization    of the yeast (1->6)-beta-glucan biosynthetic components, Kre6p and    Sknlp, and genetic interactions between the PKC1 pathway and    extracellular matrix assembly. J Cell Biol. 127(2):567-79.-   Saito, K., Yamazaki, M., Kaneko, H., Murakoshi, I., Fukuda, Y., and    van Montagu, M. (1991). Tissue-specific and stress-enhancing    expression of the T R promoter for mannopine synthase in transgenic    medicinal plants. Planta 184, 40-46.-   Schmidt K., Heberle B., Kurrasch J., Nehls R., Stahl D. J. (2004)    Suppression of phenylalanine ammonia lyase expression in sugar beet    by the fungal pathogen Cercospora beticola is mediated at the core    promoter of the gene. Plant Mol. Biol., 55: 835-852.-   Stahl D. J., Kloos, D. U., and Hehl, R. (2004). A sugar beet    chlorophyll a/b binding protein void of G-box like elements confer    strong and leaf specific reporter gene expression in transgenic    sugar beet. BMC Biotechnology 4; 31: 12-   Vancanneyt G., Schmidt R., O'Connor-Sanchez A., Willmitzer L.,    Rocha-Sosa M. (1990) Construction of an intron-containing marker    gene: splicing of the intron in transgenic plants and its use in    monitoring early events in Agrobacterium-mediated plant    transformation. Mol Gen Genet. 220(2):245-50-   Van West P, Kamoun S, van 't Klooster J W, Govers F (1999)    Internuclear gene silencing in Phytophthora infestans. Mol Cell.    March; 3(3):339-48.-   Wang Y, Dou D, Wang X, Li A, Sheng Y, Hua C, Cheng B, Chen X, Zheng    X, Wang Y (2009) The PsCZF1 gene encoding a C2H2 zinc finger protein    is required for growth, development and pathogenesis in Phytophthora    sojae. Microb Pathog. 47(2):78-86.-   Wang Y, Li A, Wang X, Zhang X, Zhao W, Dou D, Zheng X, Wang Y (2010)    GPR11, a putative seven-transmembrane G protein-coupled receptor,    controls zoospore development and virulence of Phytophthora sojae.    Eukaryot Cell 9(2):242-50.-   Yin C, Jurgenson J E, Hulbert S H (2011) Development of a    Host-Induced RNAi System in the Wheat Stripe Rust Fungus Puccinia    striiformis f. sp. Tritici. MPMI 24(5): 554-561.    doi:10.1094/MPMI-10-10-0229. © 2011 The American Phytopathological    Society-   Zhang M, Wang 0, Xu K, Meng Y, Quan J, et al. (2011) Production of    dsRNA Sequences in the Host Plant Is Not Sufficient to Initiate Gene    Silencing in theColonizing Oomyzete Pathogen Phytophthora    parasitica. PLoS ONE 6(11): e28114.-   EP 1716238 (Bayer S.A.S.) METHOD FOR MODIFYING GENE EXPRESSION OF A    PHYTOPATHOGENIC FUNGUS-   WO 2006/070227 (Devgen N.V.) METHOD FOR DOWN-REGULATING GENE    EXPRESSION IN FUNGI-   WO 2009/112270 (Leibniz-Institut für Pflanzengenetik and    Kulturpflanzenforschung) METHOD FOR CREATING BROAD-SPECTRUM    RESISTANCE TO FUNGI IN TRANSGENIC PLANTS-   US 2010/0257634 (Venganza Inc.) BIOASSAY FOR GENE SILENCING    CONSTRUCTS-   WO 2006/047495 (Venganza Inc.) METHODS AND MATERIALS FOR CONFERRING    RESISTANCE TO PESTS AND PATHOGENS OF PLANTS

1-10. (canceled)
 11. A transgenic plant of the species Solanum tuberosumor a part thereof comprising a stably integrated first double-strandedDNA and a stably integrated second double-stranded DNA, wherein thenucleotide sequences of the coding strands of the first and second DNAare reverse complements of each other, so that a transcript of the firstDNA and a transcript of the second DNA are capable of hybridizing toform a double-stranded RNA and whereby the plant expressing saiddouble-stranded RNA is resistant to an oomycete of the genusPhytophthora, said first DNA characterized in that its coding strandcomprises: (a) at least 15 successive nucleotides of a nucleotidesequence selected from the group consisting of SEQ ID NOS: 1-43, or (b)a nucleotide sequence which is complementary to at least 15 successivenucleotides of a nucleotide sequence selected from the group consistingof SEQ ID NOS: 1-43, or (c) a nucleotide sequence which hybridizes understringent conditions to at least 15 successive nucleotides of anucleotide sequence selected from the group consisting of SEQ ID NOS:1-43 or to a nucleotide sequence which is complementary to at least 15successive nucleotides of a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1-43.
 12. The transgenic plant or a partthereof of claim 11, wherein the coding strand of the first DNAcomprises: (a) a nucleotide sequence selected from the group consistingof SEQ ID NOS: 1-43, or (b) a nucleotide sequence which is complementaryto a nucleotide sequence selected from the group consisting of SEQ IDNOS: 1-43, or (c) a nucleotide sequence which hybridizes under stringentconditions to the nucleotide sequence selected from the group consistingof SEQ ID NOS: 1-43 or to a nucleotide sequence which is complementaryto a nucleotide sequence selected from the group consisting of SEQ IDNOS: 1-43.
 13. The transgenic plant or a part thereof of claim 11,characterized in that the plant or a part thereof exhibits a resistanceto Phytophthora infestans.
 14. The transgenic plant or a part thereof ofclaim 11, characterized in that the double-stranded RNA is miRNA orsiRNA.
 15. The transgenic plant or a part thereof of claim 11,characterized in that the first DNA and the second DNA are operativelylinked to at least one promoter.
 16. The transgenic plant or a partthereof of claim 11, characterized in that the first DNA and the secondDNA are under the control of the same promoter and are separated by anintron.
 17. The part of the transgenic plant of claim 11, characterizedin that it is a seed or a cell.
 18. The transgenic plant or a partthereof of claim 11, wherein the first and second DNA are completereverse complements of each other.
 19. The transgenic plant or a partthereof of claim 11, wherein the first and second DNA are partialreverse complements of each other.
 20. A method for producing atransgenic plant of the species Solanum tuberosum which plant exhibits aresistance to an oomycete of the genus Phytophthora, comprising thefollowing steps: (i) producing a transformed first parent plantcomprising a first double-stranded DNA which is stably integrated intothe genome of said first parent plant and which has a coding strandwhich comprises: (a) at least 15 successive nucleotides of a nucleotidesequence selected from the group consisting of SEQ ID NOS: 1-43, or (b)a nucleotide sequence which is complementary to at least 15 successivenucleotides of a nucleotide sequence selected from the group consistingof SEQ ID NOS: 1-43, or (c) a nucleotide sequence which hybridizes understringent conditions to at least 15 successive nucleotides of anucleotide sequence selected from the group consisting of SEQ ID NOS:1-43 or to a nucleotide sequence which is complementary to at least 15successive nucleotides of a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1-43; (ii) producing a transformed secondparent plant comprising a second double-stranded DNA which is stablyintegrated into the genome of said second parent plant, wherein thenucleotide sequences of the coding strands of the first and second DNAare reverse complements of each other, so that a transcript of the firstDNA and a transcript of the second DNA are capable of hybridizing toform a double-stranded RNA and whereby the plant expressing saiddouble-stranded RNA becomes resistant to an oomycete of the genusPhytophthora; (iii) crossing the first parent plant with the secondparent plant; (iv) selecting a plant in the genome of which both thefirst double-stranded DNA and the second double-stranded DNA have beenstably integrated.
 21. The method of claim 20, wherein the coding strandof the first DNA comprises: (a) a nucleotide sequence selected from thegroup consisting of SEQ ID NOS: 1-43, or (b) a nucleotide sequence whichis complementary to a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1-43, or (c) a nucleotide sequence whichhybridizes under stringent conditions to the nucleotide sequenceselected from the group consisting of SEQ ID NOS: 1-43 or to anucleotide sequence which is complementary to a nucleotide sequenceselected from the group consisting of SEQ ID NOS: 1-43.
 22. The methodof claim 20, characterized in that the resistance is resistance toPhytophthora infestans.
 23. The method of claim 20, characterized inthat the double-stranded RNA is miRNA or siRNA.
 24. The method of claim20, wherein the first and second DNA are complete reverse complements ofeach other.
 25. The method of claim 20, wherein the first and second DNAare complete reverse complements of each other.
 26. A composition forexternal application to plants comprising a double-stranded RNA, whereina strand of said RNA corresponds to a transcript of a double-strandedDNA comprising a coding strand which comprises: (a) at least 15successive nucleotides of a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 1-43, or (b) a nucleotide sequence which iscomplementary to at least 15 successive nucleotides of a nucleotidesequence selected from the group consisting of SEQ ID NOS: 1-43, or (c)a nucleotide sequence which hybridizes under stringent conditions to atleast 15 successive nucleotides of a nucleotide sequence selected fromthe group consisting of SEQ ID NOS: 1-43 or to a nucleotide sequencewhich is complementary to at least 15 successive nucleotides of anucleotide sequence selected from the group consisting of SEQ ID NOS:1-43.
 27. The composition of claim 26, wherein the coding strandcomprises: (a) a nucleotide sequence selected from the group consistingof SEQ ID NOS: 1-43, or (b) a nucleotide sequence which is complementaryto a nucleotide sequence selected from the group consisting of SEQ IDNOS: 1-43, or (c) a nucleotide sequence which hybridizes under stringentconditions to the nucleotide sequence selected from the group consistingof SEQ ID NOS: 1-43 or to a nucleotide sequence which is complementaryto a nucleotide sequence selected from the group consisting of SEQ IDNOS: 1-43.
 28. A method for conferring resistance against an oomycete ofthe genus Phytophthora in a plant or a plant part, said methodcomprising administering to said plant or plant part the composition ofclaim
 26. 29. The method of claim 28, wherein the oomycete of the genusPhytophthora is Phytophthora infestans.
 30. The method of claim 28,wherein the plant part is a seed or a cell.
 31. The method of claim 28,wherein the plant is from the species Solanum tuberosum.